RESEARCH ARTICLE 3561
Development 139, 3561-3571 (2012) doi:10.1242/dev.079491
© 2012. Published by The Company of Biologists Ltd
AIP1 acts with cofilin to control actin dynamics during
epithelial morphogenesis
Dandan Chu1, Hanshuang Pan1, Ping Wan1, Jing Wu1, Jun Luo1, Hong Zhu1 and Jiong Chen1,2,*
SUMMARY
During epithelial morphogenesis, cells not only maintain tight adhesion for epithelial integrity but also allow dynamic intercellular
movement to take place within cell sheets. How these seemingly opposing processes are coordinated is not well understood. Here,
we report that the actin disassembly factors AIP1 and cofilin are required for remodeling of adherens junctions (AJs) during
ommatidial precluster formation in Drosophila eye epithelium, a highly stereotyped cell rearrangement process which we describe
in detail in our live imaging study. AIP1 is enriched together with F-actin in the apical region of preclusters, whereas cofilin displays
a diffuse and uniform localization pattern. Cofilin overexpression completely rescues AJ remodeling defects caused by AIP1 loss of
function, and cofilin physically interacts with AIP1. Pharmacological reduction of actin turnover results in similar AJ remodeling
defects and decreased turnover of E-cadherin, which also results from AIP1 deficiency, whereas an F-actin-destabilizing drug affects
AJ maintenance and epithelial integrity. Together with other data on actin polymerization, our results suggest that AIP1 enhances
cofilin-mediated actin disassembly in the apical region of precluster cells to promote remodeling of AJs and thus intercellular
movement, but also that robust actin polymerization promotes AJ general adhesion and integrity during the remodeling process.
INTRODUCTION
Adherens junction (AJ) remodeling is crucial for the dynamic
rearrangement of cells within cell sheets during epithelial
morphogenesis, which occurs widely during developmental and
physiological processes (Nishimura and Takeichi, 2009).
Cadherins, such as E-cadherin (E-cad), together with -catenin and
-catenin comprise the core of the AJ. The actin cytoskeleton is
thought to provide the mechanical support for AJs to join
neighboring cells together as a tight layer, but recent studies have
increasingly shown that the nature of the actin network underlying
AJs is more dynamic than previously thought (Harris and Tepass,
2010). During epithelial remodeling in Drosophila embryos, the
actin network turns over rapidly and is proposed to promote the
lateral mobility of microdomains of homophilic E-cad clusters
(sites of strong intercellular adhesion), allowing AJ remodeling to
take place (Cavey et al., 2008).
Biochemical studies have established cofilin as a major factor in
promoting actin dynamics, which is achieved through its dual
function of filament severing and monomeric actin (G-actin)
dissociation from the pointed ends (Bamburg, 1999). The
depolymerizing function of cofilin was reported to promote a high
rate of actin treadmilling, which occurs when G-actin
depolymerized from the pointed ends of the filaments is
continuously polymerized onto their barbed ends, allowing
continuous and robust actin polymerization to take place in
dynamic cellular regions including the lamellipodia, growth cones
and sites of endocytosis (Bamburg, 1999; Wang et al., 2007). AIP1
1
Model Animal Research Center, and MOE Key Laboratory of Model Animals for
Disease Study, Nanjing University, Nanjing 210061, China. 2Zhejiang Provincial Key
Lab for Technology and Application of Model Organisms, School of Life Sciences,
Wenzhou Medical College, Wenzhou 325035, China.
*Author for correspondence ([email protected])
Accepted 9 July 2012
(Actin interacting protein 1) was identified as a major co-factor that
collaborates with cofilin to disassemble F-actin. AIP1 can bind to
the cofilin–F-actin complex and strongly enhance the severing
activity of cofilin on actin filaments by capping the barbed ends of
the severed filaments, resulting in acceleration of actin
depolymerization (Ono, 2003). Evidence from microscopy
indicates that AIP1 can actively disassemble cofilin-bound actin
filaments (Ono, 2003).
Both cofilin (17 kDa) and AIP1 (67 kDa) are highly conserved
across all eukaryotes, from yeast and plants to mammals. Our
previous studies showed that cofilin is genetically required for cell
migration in the Drosophila ovary and that it promotes
lamellipodial protrusions (Chen et al., 2001; Zhang et al., 2011).
Other genetic studies have shown that cofilin is essential for a
variety of cellular and developmental processes requiring strong
actin dynamics, including cytokinesis, axon growth and
endocytosis (Bamburg, 1999; Ono, 2003). Functional analyses of
AIP1 in model organisms has revealed its roles in actin-based
processes including body wall muscle formation in C. elegans,
neutrophil response in mouse, cytokinesis, phagocytosis and cell
motility in Dictyostelium (Kile et al., 2007; Ono, 2003). However,
whether AIP1 or cofilin is essential for AJ remodeling in the
epithelia is unclear. Recently, point mutations in flare (flr), the gene
that encodes Drosophila AIP1, have been reported to disrupt planar
cell polarity (PCP) and the morphology of wing hairs (Ren et al.,
2007), but no specific AJ defects were directly attributed to
disruption of AIP1 function in the wing disc epithelium.
In this study, we show that AIP1 and cofilin are essential for AJ
remodeling during morphogenesis of eye epithelia in Drosophila
larvae. Treatment with drugs that reduce actin disassembly reveals
the important role of actin turnover in epithelial morphogenesis,
including the process of AJ remodeling during ommatidial
precluster formation. Here, we also present a live imaging study
that describes in detail the process of AJ remodeling in precluster
formation, which is affected in the flr mutant. Furthermore,
enrichment of AIP1 together with F-actin in the apical region of
DEVELOPMENT
KEY WORDS: AIP1 (Flare), Actin dynamics, Adherens junction remodeling, Epithelial morphogenesis, Cofilin (Twinstar)
Development 139 (19)
3562 RESEARCH ARTICLE
MATERIALS AND METHODS
Drosophila genetics
Fly strains are described in supplementary material Table S1. Flies were
raised on standard cornmeal and sucrose media, and all crosses were
performed at 25°C except for temperature shift experiments.
Immunohistochemistry and microscopy
Eye discs of third instar larvae were dissected in 1⫻PBS, fixed in 7.4%
formaldehyde for 30 minutes, blocked for 30 minutes in PBT (0.3%
Triton X-100 in PBS) containing 10% normal goat serum, and incubated
overnight at 4°C in primary antibodies as follows: rat anti-Elav [1:100,
Developmental Studies Hybridoma Bank (DSHB)], mouse anti-Prospero
(1:50, DSHB), mouse anti-Armadillo (1:100, DSHB), mouse anti-Dlg1
(1:100, DSHB), rabbit anti-PKC (1:500, Santa Cruz), rabbit anti-galactosidase (1:2000, Rockland), rabbit anti-cofilin (1:10,000; gift of
James Bamburg; Shaw et al., 2004), rat anti-E-cad (DCAD2, 1:50,
DSHB), rat anti--catenin (DCAT-1, 1:100, DSHB), mouse anti-spectrin (3A9, 1:50, DSHB), goat anti-Arp2 [sc-11968, 1:10, Santa Cruz
(Rogers et al., 2003)] and mouse anti-profilin (CHI 1J, 1:10, DSHB).
Cy3- and Cy5-conjugated secondary antibodies (1:250, Jackson) were
used. F-actin was labeled by Rhodamine phalloidin (1:400, Sigma) for
30 minutes. Single confocal sections and z-stacks were acquired using a
Leica TCS SL or Olympus FV1000 confocal microscope; cross-section
reconstructions were performed using Leica or Olympus software.
Western blotting and immunoprecipitation
Second instar larvae of various flr mutants were collected for western
analysis using standard protocols. Protein extracts were analyzed by
immunoblotting with the following primary antibodies: rabbit anti-AIP1
(1:5000, raised against full-length Drosophila AIP1 protein), mouse antiactin (1:10,000, Millipore), mouse anti-GFP (1:1000, Roche) and rabbit
anti-cofilin (1:10,000; Shaw et al., 2004). Secondary antibodies were HRP
conjugated (Jackson). The intensity of protein bands was quantified using
ImageJ (NIH). For the immunoprecipitation assay, protein extracts from
third instar larvae were incubated with 20 l anti-GFP agarose beads
(MBL, Japan) for 8 hours at 4°C; lysates and immunoprecipitates were
then immunoblotted.
Time-lapse imaging and fluorescence recovery after
photobleaching (FRAP)
Third instar larval eye discs were dissected with optic lobes attached in
Schneider’s medium cocktail (Prasad et al., 2007) and mounted on a
Lumox dish (Sigma) for live imaging with an Olympus FV1000 confocal
microscope.
For the FRAP experiment, eye discs were dissected in Schneider’s
medium cocktail (without insulin), incubated in the medium containing 10
M Jasp (dissolved in DMSO, Invitrogen) or an equivalent volume of
DMSO for 10 minutes, and then mounted on a Lumox dish. Photo
bleaching was performed in a region of interest (ROI; typically ~0.7 m ⫻
0.5 m) using a 488-nm laser at 100% for ~250 mseconds. FRAP images
were collected (60⫻ oil-immersion Olympus confocal lens, NA1.42; a
single confocal section collected for each time point) at 10 seconds/pixel
and imported into ImageJ for intensity measurement. The fluorescence
intensity (FI) of the ROI at each time point was normalized against the FI
of the ROI before bleaching and expressed as a percentage of initial FI
[Inormalized (t)] according to the following formula (Seabrooke et al., 2010):
Inormalized (t)  (AIprebleach–BG) (FIt–BG)/(AIt–BG) (FIprebleach–BG),
where AI is the average FI of the AJs of bleached cells including the ROI,
FI is the FI of the ROI, and BG is the FI of the background region outside
of the ROI and AJs. A recovery curve was fitted for each dataset using the
following equation (Weisswange et al., 2009):
Ifitted (t)  (ImaxImin) (1–e–kt),
where Ifitted(t) is the fitted percentage of the initial FI at time t, Imax is the
maximum Inormalized, Imin is the minimum Inormalized, and k is the rate constant
of recovery. Individual half times of recovery were calculated from
individual fitted curves and were statistically compared (Student’s t-test)
between DMSO control (n15) and flr mutant (n18) or between DMSOtreated control and Jasp-treated (n15) preclusters. The final curves were
then plotted using the mean value for each time point, and the s.e.m.
calculated for each time point (see also supplementary material Fig. S7).
Drug treatment
Eye discs were dissected and cultured in Schneider’s medium cocktail
(without insulin) containing 2 M Jasp, 2 M Lat-A (Invitrogen), or an
equivalent volume of DMSO for 2 hours, and then fixed as described above.
RESULTS
AIP1 is required for eye development
The crystalline lattice pattern of 750-800 ommatidia in the
Drosophila adult compound eye is laid down at the late larval (third
instar) stage, when equally spaced columns of ommatidial
rudiments are formed in the wake of the morphogenetic furrow
(MF) passing from the posterior end of the eye disc epithelium to
the anterior end. Anterior to the MF, cells remain undifferentiated
and unpatterned. Posterior to the MF, active morphogenesis and
cell fate specification take place to form columns of equally spaced
clusters of photoreceptor progenitors, namely ommatidial
preclusters immediately posterior to the MF and the more
differentiated ommatidial clusters further posterior to the MF
(Wolff and Ready, 1991). This early phase of eye morphogenesis
is crucial for the development and patterning of the adult eye, as its
disruption can cause perturbation in cluster morphology and
spacing, resulting in a ‘rough’ adult eye. To address whether AIP1
is required for epithelial morphogenesis during eye development,
we performed genetic mosaic experiments using the FRT-FLP
system (Chen et al., 2005; Xu and Rubin, 1993), and showed that
lethal mutations in flr induced by P-elements resulted in a strong
rough phenotype in the mosaic eyes of adult flies (Fig. 1A⬙;
supplementary material Fig. S1). The three P-element alleles we
tested were hypomorphic and gave a lethal phenotype at 25°C
except for flrBG, which produced homozygous escapers at 18°C and
also displayed rough eyes (Fig. 1A⬘). A genomic fragment
encompassing the flr locus was able to rescue both the lethality and
rough eye phenotype for all three alleles (Fig. 1D; supplementary
material Fig. S1), indicating they were bona fide flr mutations.
Lastly, expressing flr RNAi in the eye disc in flip-out clones or by
ey-Gal4 produced similar adult and larval eye defects (see Fig. 7F⬘;
supplementary material Fig. S1D).
To determine the cause of the rough phenotype, we first
examined the organization of ommatidial clusters in the mosaic
larval eye epithelium. Immunostaining for Elav, a nuclear marker
for photoreceptor (R) cell fate, showed that cells in flr mutant
clones did acquire R fates but were disorganized in their
arrangement within each ommatidial cluster, as compared with
adjacent heterozygous (wild-type) tissues (Fig. 1B⬘,C⬘).
Consequently, spacing between mutant clusters was more irregular
than between wild-type clusters. Labeling by Svp--gal (the R3,
R4, R1, R6 marker) and antibody against Prospero (marking R7
and the 4 cone cells) further confirmed that most of the mutant
clusters displayed the proper cell fates but were disorganized (Fig.
1B⬙,C⬙). The disorganization was not caused by excessive
apoptosis (supplementary material Fig. S2H,H⬘). Lastly, the nuclear
Elav staining in the mutant R cells appeared less intense than in
neighboring wild-type R cells (Fig. 1B⬘), which was likely to be
due to the more basal positioning of mutant nuclei (supplementary
DEVELOPMENT
these preclusters suggests that AIP1 specifically promotes an
environment of strong actin dynamics to allow fast AJ remodeling
to take place during eye epithelial morphogenesis.
Actin dynamics and adherens junction remodeling
RESEARCH ARTICLE 3563
material Fig. S1F-F⵮). Together, these results suggest that the effect
of flr mutation on R cell differentiation is indirect and is likely a
consequence of disruption in the normal epithelial morphology of
R cells.
Disrupting AIP1 function affects actin
cytoskeleton and AJs in preclusters and clusters
To reveal the underlying cause of cluster disorganization, we
examined the actin cytoskeleton and epithelial morphology.
Immunostaining using antibodies against Armadillo (Arm; catenin), E-cad (Shotgun – FlyBase) and -catenin showed that
AJs were significantly disrupted in the flr mutant clusters (Fig. 2AB⬙; supplementary material Fig. S2A-F⬘). In wild type, AJs within
a typical mature eight-cell (R1-8) cluster usually display six or
seven tightly connected Arm-stained lines in the apical region,
which is highly constricted (Fig. 2B⬘). In flr mutant clusters, these
lines of strong staining were dramatically disrupted. Arm or catenin staining appeared disjointed, diffuse and sometimes
punctate, and the apical region was no longer constricted (Fig.
2B⬘,E⬘; supplementary material Fig. S2C⬘,F⬘). To determine
whether the flr mutation primarily affects AJs and not other
junctional regions, we examined the distribution of the apical
marker aPKC (part of the aPKC-Par3-Par6 complex) and the lateral
(septate) junction marker Dlg1. In flr mutant clones, a cross-section
reconstructed from a confocal z-stack showed that Dlg1 was
distributed along the apical-basal axis normally (Fig. 2D⬘). Within
mutant clusters, aPKC was localized at a region more apical than
the AJ region, similar to in wild type (Fig. 2E⬙,E⵮), but Arm
staining in mutant clusters often appeared to localize slightly more
basally than in wild type (Fig. 2E-E⵮). Furthermore, a single
confocal section taken in the apical plane showed that in both
mutant and wild-type clusters aPKC staining appeared as a cap
over the AJs of each cluster (Fig. 2E⬙), with the reduced staining
intensity in the mutant clusters a likely result of the expanded
apical surface. Together, these results indicate that flr mutation
primarily affects AJ morphogenesis in the ommatidial clusters.
To determine whether the dramatic AJ disorganization in the
ommatidial clusters arose from earlier AJ defects, we examined the
process of precluster formation, as occurs in and immediately
posterior to the MF. Phalloidin staining showed that F-actin levels
were increased across the mutant tissue posterior to the MF, with
the increase more prominent in preclusters and clusters (Fig. 2C,C⬘,
Fig. 3A⬘; supplementary material Fig. S2B⬘,D⬘). This is consistent
with the proposed role of AIP1 as a co-factor of cofilin in
DEVELOPMENT
Fig. 1. flr mutations result in defects
in the adult eye and larval eye disc.
(A-A⬙) Scanning electron micrographs of
adult eyes from wild-type (w1118),
homozygous flrBG and flrEY-P2 mosaic
Drosophila. (B-B⵮) Confocal micrograph
of an flrEY-P2 mosaic larval eye disc
showing Elav and Prospero staining.
Clones are marked by the absence of GFP,
and clones of significant size are outlined
(white dotted line). (C-C⵮) Magnified view
of a mutant clone showing the presence
of R3, R4, R1 and R6 in disorganized
ommatidial clusters, as revealed by the
merge of Elav and Svp--gal stainings.
White box outlines a wild-type cluster and
yellow box outlines a mutant cluster.
(D)Structure of the flr locus showing
three P-element insertions in the 5⬘ UTR
region and a GFP trap insertion in the first
intron. A black line indicates the region
encompassed by the genomic rescue
fragment G2A, and red blocks indicate
the exonic sequence used in the RNAi
construct. (E)A western blot stained with
antibody against Drosophila AIP1 shows
significant reduction of AIP1 levels in the
larvae of flrEY-P1, flrEY-P2 and flrBG, as
compared with the wild type (w1118).
(F)Quantification of data in E. (G)Western
blot analysis of endogenous AIP1 and
AIP1-GFP in w1118 and flrGFP larvae.
(H)w1118 and flrGFP larval extracts were
immunoprecipitated with anti-GFP-bound
agarose beads and then immunoblotted
with anti-cofilin. Scale bars: 100m in AA⬙; 20m in B-C⵮.
3564 RESEARCH ARTICLE
Development 139 (19)
disassembling F-actin. A cross-section showed that the increase in
F-actin staining was largely confined to the apical and sup-apical
region that includes AJs (Fig. 2C⬘), suggesting that AIP1 acts in
this region of preclusters and clusters to regulate actin dynamics.
By contrast, F-actin levels and cell shape remained unaffected in
mutant clones anterior to the MF (Fig. 2C; supplementary material
Fig. S2I-I⵮), suggesting that AIP1 function is required in regions
of the eye epithelium that are undergoing more active
morphogenesis (i.e. posterior to the MF). However, since the flr
alleles used were all hypomorphic, we cannot rule out the
possibility that AIP1 might still be required for morphogenesis of
anterior tissue.
Upon closer examination of the MF-proximal area, we found
clear F-actin defects in three to four columns of preclusters at
various stages of formation (Fig. 3A-C⬙). Arm staining revealed
that these preclusters exhibited disorganization in the AJ pattern
compared with wild type (Fig. 2A-A⬙, Fig. 3B-F⬙; supplementary
material Fig. S2E-E⬙). AJs within a typical wild-type precluster
(containing the core of R8, the R2 R5 pair and the R3 R4 pair)
usually appeared as four strongly stained lines between the five
cells with future R fates, along with lines of lesser intensity
between non-R cells (Fig. 2A⬘, Fig. 3B⬘). By contrast, in flr mutant
preclusters, marked by the significantly increased cortical F-actin
staining, these four Arm-stained lines between the five R cells had
lost some of their staining intensity and became indistinguishable
from AJs between other non-R cells (Fig. 2A⬘, Fig. 3C-C⬙).
Together, these data show that the actin disassembly function of
AIP1 is required in epithelial cells undergoing morphogenesis
posterior to the MF and that its activity is required to remodel AJs
within the preclusters so as to achieve a polarized AJ distribution
between the five R cells.
Live imaging of eye discs reveals dynamic
remodeling of AJs in and posterior to the MF
Previous work by Wolff and Ready described various stages of
precluster formation in the MF based on fixed samples (Fig. 3H)
(Wolff and Ready, 1991). Recently, Escudero and co-workers
performed live imaging analysis on the dynamic distribution of
myosin II in the MF and in the preclusters, and they have traced
precluster rotation (after preclusters have formed) in real time using
Arm-GFP (Escudero et al., 2007). To our knowledge, no study has
yet been done to specifically describe in detail how AJs are
remodeled in real time during precluster formation. To better
understand this dynamic morphogenetic event and the AJ
remodeling defects resulting from flr mutation, we performed live
imaging of eye discs using an Arm-GFP transgene to track the
dynamic changes in AJs that occur during normal precluster
formation.
Late third instar larval eye discs can be cultured in medium
for 3 hours without obvious morphological defects, and ArmGFP signals were indistinguishable from Arm staining in fixed
tissues (Fig. 3B⬘,G). Live imaging revealed that a precluster
comprising five R cells formed through a stereotyped process
(Fig. 3I,I⬘; supplementary material Movie 1). The first visible
sign of precluster formation was in the form of a large ‘rosette’
comprising an outer ring of ~13-16 cells and an inner core of
about four to six cells, with the progenitors of five R cells always
at the posterior end of the ring. At this initial stage, AJs between
any two adjacent cells within the rosette displayed a similarly
strong Arm-GFP signal, except that the AJs at the posterior end
showed a slightly more intense signal, suggesting that the five R
progenitor cells might have begun their specification at this
stage. Sixty minutes later, the rosette was transformed into a
DEVELOPMENT
Fig. 2. flr mutation causes specific AJ defects in the ommatidial preclusters and clusters. (A-A⬙) In a large flrEY-P2 mutant clone spanning the
MF, the distinct AJ pattern marked by Arm is disrupted in the preclusters immediately posterior to the MF. (B-B⬙) Further posterior to the MF, more
mature clusters within mutant clones display dramatic disruption of the Arm staining pattern in the AJs. Clones are marked by absence of GFP.
(C,C⬘) A wide mutant clone across the MF (outlined by the white dotted line) displays a substantial increase in F-actin levels (stained by phalloidin)
only posterior to the MF; cross-sectional view is to the right (the yellow dotted line marks the position of the cross-section). Apical is to the left and
basal to the right for all cross-sections. (D,D⬘) The distribution of Dlg1 appears normal in mutant clones of a mosaic eye disc. The cross-section
covers only the lateral part. (E-E⵮) Distribution of Arm but not of aPKC is grossly affected in a mutant clone. The cross-section is focused on the
apical part. Yellow brackets indicate two mutant clusters and the white bracket marks an adjacent wild-type cluster. Scale bars: 5m in A-B⬙,E-E⵮;
50m in C-D⬘.
Actin dynamics and adherens junction remodeling
RESEARCH ARTICLE 3565
more elongated cluster by a series of cell shape changes
accompanied by AJ remodeling, resulting in a distinct arc of
about six cells (including five R cells) at the posterior end.
Notably, the Arm-GFP signal within the arc was significantly
stronger than outside the arc. After 90-150 minutes, the arc
underwent extensive intercellular movement so that cells at
either end of the arc that previously did not contact each other
could now form AJs between them. As shown in Fig. 3I (at 60
minutes), the R3 and R4 precursors were originally far apart at
either end of the arc. But by 150 minutes, R3 and R4 had
established an AJ between them, such that the five R precursor
cells could form a distinct and compact cluster with the trio of
R8, R2 and R5 at the posterior and the R3 and R4 pair at the
anterior. We refer to such a cluster at the end of precluster
formation as a five-cell precluster.
In flrEY-P2 mutant clones, cells at the posterior position of a
rosette often failed to remodel their AJs to first form a distinct arc
and then a compact five-cell precluster (Fig. 3C-F⬙, Fig. 4).
Interestingly, the arc in the wild-type cluster always proceeded
through a transitory ‘six-cell rosette’ form (16 clusters examined;
Fig. 3I, 120 minutes), in which the non-adjacent R3 and R4
remodeled their AJs with a common non-R cell, called the M cell
(the ‘mystery’ cell, from older literature) (Wolff and Ready, 1991),
to bring themselves into contact with each other, resulting in six
cells (five R cells and one M cell) sharing a common AJ ‘vertex’.
In mutant tissues, such an intermediate form with strong Arm
staining in the vertex was rarely observed (Fig. 3E⬘,F⬘).
Furthermore, the general AJ organization in mutant cells, as shown
by the continuous E-cad or Arm staining outlining each cell (Fig.
3F-F⬙; supplementary material Fig. S2E-E⬙), appeared to be
DEVELOPMENT
Fig. 3. Precluster formation as revealed by live imaging and fixed tissue staining. (A,A⬘) Low magnification view of a mosaic eye disc
displaying precluster formation; flrEY-P2 mutant clones are outlined (white dotted line). (B-C⬙) High magnification view of wild-type tissue (B-B⬙) and a
nearby mutant clone (C-C⬙) as outlined by the blue and yellow boxes, respectively, in A⬘. A strong increase in F-actin levels occurs in the precluster
primordia and appears to be cortical, underlying Arm-labeled AJs (C⬙). 1, 2, 3 indicate the column in which each precluster is located.
(D-F⬙) Enlarged view of wild-type (E-E⬙) and mutant (F-F⬙) preclusters as numbered in B⬘ and C⬘, respectively. (D-D⬙) Schematic interpretations of
wild-type AJ staining in E-E⬙ showing progressive stages during precluster formation. (G)Low magnification view of the MF and preclusters of a
cultured eye disc during live imaging; Arm-GFP provides labeling of AJs. A, anterior direction; E, equatorial direction. (H) Diagram of precluster
formation based on lead sulfide staining of the membrane by Wolff and Ready (Wolff and Ready, 1991). A large rosette (column 0) is shown
emerging from the MF and developing into an arc of various shapes (in columns 1, 2 and 3), finally forming a five-cell precluster (column 4). (I,I⬘) A
time-lapse series of images showing dynamic remodeling of AJs during the formation of a five-cell precluster. A large rosette (from the yellow box
in G) and subsequent clusters of cells derived from it are outlined with yellow dotted lines. Schematic interpretations of the time-lapse series are
shown beneath. The green line or dot indicates the AJ contact between R3 and R4. Scale bars: 10m in A-C⬙; 1m in E-F⬙,I; 5m in G.
3566 RESEARCH ARTICLE
Development 139 (19)
normal, unlike the highly disjointed AJs observed in the more
mature ommatidial clusters (Fig. 2B⬘). Finally, live imaging of
mosaic eye discs containing flrEY-P2 mutant clones clearly showed
defects in AJ (visualized with E-cad-GFP) remodeling in mutant
preclusters, leading to a failure to form the six-cell rosette and fivecell precluster (Fig. 4; supplementary material Movie 2). There was
inadequate intercellular movement to bring R3, R4 and M cells
together as a six-cell rosette in the mutant (Fig. 4B⬘) in contrast to
the wild type (Fig. 4A⬘). Taken together, these data indicate that
disruption of AIP1 function affects remodeling but not general
maintenance of AJs during precluster formation.
Disrupting actin turnover affects AJ remodeling
posterior to the MF
Because AIP1 was thought to promote actin turnover and its loss
of function results in elevated levels of F-actin in both preclusters
and clusters as shown above, drugs that block turnover of F-actin
could in theory phenocopy the AJ remodeling defects in precluster
formation seen in the mutant tissues. To test this, we treated wildtype eye discs with jasplakinolide (Jasp), a drug that binds to and
prevents F-actin from depolymerizing, thus acting as an inhibitor
of actin turnover (Bubb et al., 1994). Eye discs cultured in control
medium for 2 hours displayed normal MF, precluster and cluster
morphologies, as shown by the E-cad-GFP signal (Fig. 5A,A⬘). By
contrast, eye discs cultured in 2 M Jasp for 2 hours exhibited
strong AJ remodeling defects within and posterior to the MF,
similar to those observed in the flr mutant clones (Fig. 5C,C⬘). In
a control eye disc, different stages of precluster formation were
clearly seen in four columns of equally spaced clusters behind the
MF as rosettes, arcs or five-cell preclusters (Fig. 5A⬘). But in a
Jasp-treated eye disc, these distinct forms were absent and few
preclusters displayed the strong and asymmetric E-cad-GFP
distribution pattern characteristic of a developing wild-type
precluster (Fig. 5C⬘). Interestingly, both Jasp treatment and flr
mutation caused a significant increase in apical surface area in the
five R cells of preclusters as compared with the control
(supplementary material Fig. S3K). Together, these data indicate
that blocking actin turnover affects various AJ remodeling
processes, including that during precluster formation, similar to the
flr mutant phenotype (Fig. 2A⬘), further confirming that the AIP1
actin turnover function plays essential roles in AJ remodeling.
Treatment with the F-actin-destabilizing drug latrunculin A (LatA), which sequesters G-actin and thus induces rapid disassembly
of F-actin, resulted in severe disruption of AJs within and posterior
to the MF (Fig. 5B,B⬘). Eye discs cultured in 2 M Lat-A for 2
hours exhibited a strong defect in the general organization of AJs,
E-cad-GFP signals appeared to be mostly diffuse and disorganized
and this effect was more dramatic in the seven to eight columns
behind the MF (Fig. 5B), often accompanied by severe disruption
of epithelial integrity around the MF region (supplementary
material Fig. S3A). Together, these data suggest that Lat-A leads to
disruption of cell adhesion and AJ integrity, whereas reduction of
actin turnover from Jasp treatment results in insufficient AJ
remodeling and thus a lack of intercellular movement within the
epithelial sheet.
To further understand the dynamic nature of the actin
cytoskeleton underlying AJs in the eye disc, we utilized a
previously reported actin dynamics assay to label newly
polymerized F-actin (Lee et al., 2009). Following incubation of eye
discs in a high concentration (10 M) of Jasp for 10 minutes to
mask existing F-actin, immediate staining with Rhodamine
phalloidin, which competes with Jasp for F-actin binding, would
normally detect no signal (supplementary material Fig. S3A-A⬙).
If Jasp-treated eye discs were incubated in drug-free medium to
recover for a period of time, any newly polymerized F-actin could
be labeled by staining with Rhodamine phalloidin at different time
points. We found that after 90 minutes of recovery time, the four
to eight columns behind the MF comprised the first region in the
eye disc to show phalloidin staining, and the staining was most
intense in the developing preclusters (supplementary material Fig.
DEVELOPMENT
Fig. 4. Live imaging of precluster
formation in the flr mutant. Time-lapse
images showing AJ remodeling of a wild-type
rosette (A) and an flr mutant rosette (B) from
a single flr mosaic eye disc. Schematic
interpretations (A⬘,B⬘) are shown beneath.
The green dot indicates the AJ vertex of a sixcell rosette. Scale bar: 10m.
Actin dynamics and adherens junction remodeling
RESEARCH ARTICLE 3567
Fig. 5. Blocking actin turnover affects AJ remodeling and E-cad dynamics. (A-C⬘) An eye disc cultured in DMSO control (A,A⬘) shows normal
E-cad-GFP signal, whereas Lat-A (B,B⬘) or Jasp (C,C⬘) treatment each results in different AJ defects. The boxed MF-proximal regions in A-C are
enlarged in A⬘-C⬘. Arrow indicates MF. (D-G) FRAP analysis of E-cad-GFP for precluster AJs. (D-F)Colored boxes indicate the region of interest (ROI),
where photobleaching is applied and fluorescence intensity is then measured every second for 60 seconds. (G)Recovery curves for DMSO-treated,
Jasp-treated and flr mosaic (red) eye discs. For each data point the s.e.m. is shown. See also Fig. S7 in the supplementary material. Scale bars:
20m in A-C; 10m in A⬘-C⬘; 1m in D-F.
Reduced actin turnover leads to decreased E-cad
turnover within AJs of preclusters
A previous study of Drosophila embryo morphogenesis
demonstrated that homophilic E-cad clusters within the AJ
underwent lateral mobility to allow AJ remodeling to take place
(Cavey et al., 2008). Therefore, reducing actin turnover might
affect AJ remodeling in the preclusters by restricting the lateral
mobility of E-cad within AJs. To test this, we performed FRAP
analyses on the effects of flr mutation and Jasp treatment on E-cad
turnover in precluster AJs (Fig. 5G-J). AJs between any two of the
five R cells within developing preclusters at column 2 were chosen
for FRAP on control (DMSO), Jasp and flrEY-P2 mosaic eye discs.
The control eye discs displayed a robust E-cad-GFP recovery curve
with an average half-time of 21.5±0.8 seconds (n15 preclusters),
whereas significantly longer half-times were found for the Jasptreated (25.3±1.1 seconds; n15, P0.0174) and flrEY-P2 (26.4±1.4
seconds; n18, P0.0096) eye discs. These results indicate that
Jasp treatment and loss of AIP1 function slows down E-cad
turnover in precluster AJs.
Inhibition of endocytosis results in different AJ
phenotypes than AIP1 loss of function
AJ dynamics can also be regulated by clathrin-dependent
endocytosis, as shown by previous reports, and cofilin is required
for endocytosis in yeast (Bamburg, 1999; Nishimura and Takeichi,
2009). To address whether the AJ remodeling defects that result
from AIP1 deficiency or Jasp treatment are due to a defect in
endocytosis, we treated the eye disc with monodansylcadaverine
(MDC), a drug that blocks clathrin-dependent endocytosis (Schütze
et al., 1999). MDC treatment resulted in uniform elevation of Ecad levels throughout the eye disc (supplementary material Fig.
S5B,B⬘), which is consistent with its endocytosis blocking effect,
but this was never observed in flr or Jasp-treated eye discs. In
MDC-treated discs, all stages of precluster formation were still
clearly discernible except that the shapes of some preclusters
appeared slightly different from those of wild type, whereas Jasp
treatment and flr mutation both caused much more severe AJ
remodeling defects including loss of distinct arc, six-cell rosette
and five-cell precluster forms in the first four to five columns (Fig.
3D-F⬙, Fig. 5C⬘). Lastly, MDC did not result in any significant
apical surface increase, in contrast to flr or Jasp-treated eye discs
(supplementary material Fig. S3G). These results suggest that the
AJ remodeling defects caused by AIP1 loss of function or Jasp
treatment are unlikely to be due to a block in E-cad endocytosis.
AIP1 is enriched in the apical region of preclusters
whereas cofilin displays a diffuse localization
To further address the role of AIP1 in eye epithelial morphogenesis,
we examined its distribution pattern. We obtained a protein-trap
insertion allele, resulting in an N-terminal fusion of GFP to the AIP1
protein. This flrGFP allele was homozygous viable with no adult eye
defects and generated only the AIP1-GFP fusion (94 kDa) and no
endogenous AIP1 (67 kDa), as shown in a western blot using
antibodies generated against full-length Drosophila AIP1 (Fig. 1G).
DEVELOPMENT
S3C-C⬙). After longer periods of recovery, additional phalloidin
labeling could be detected in more posterior columns of ommatidial
clusters (data not shown). These data suggest that actin
polymerization is most robust in the region within or immediately
posterior to the MF, particularly within individual preclusters
undergoing morphogenesis. Lastly, when the same Jasp recovery
assay was performed on mosaic eye discs containing flrEY-P2 clones,
we found that newly polymerized F-actin levels were increased
within the mutant clones as compared with adjacent wild-type
tissues (supplementary material Fig. S3D⬘). This result suggests
that AIP1 functions to depolymerize newly polymerized F-actin,
and the accumulation of newly polymerized F-actin underscores
the importance of depolymerization during rapid actin treadmilling
in the developing preclusters and clusters.
These data indicate that AIP1-GFP can fully replace the function of
endogenous AIP1 in the homozygous flrGFP animals and can thus be
used as a reliable marker for AIP1. Indeed, immunostaining with
AIP1 antibody showed an almost identical staining pattern to that of
AIP1-GFP (supplementary material Fig. S4B-B⬙); thus, we used
flrGFP for all the distribution analyses below.
We found that anterior to the MF, AIP1 was cytoplasmic and
diffusely localized, whereas posterior to the MF its levels were
upregulated and AIP1 was enriched together with F-actin in the
apical region of individual preclusters and clusters (Fig. 6;
supplementary material Fig. S5), confirming the idea that AIP1 acts
in the apical/AJ region of precluster cells to promote actin
dynamics and AJ remodeling. Surprisingly, cofilin did not display
enrichment in the AJ region of preclusters and clusters, and its
distribution pattern posterior to the MF was much more uniform
than that of AIP1 (Fig. 6C-C⵮). This difference in distribution
patterns suggests a specific functional role of AIP1 in promoting
F-actin dynamics underlying precluster or cluster AJs. Lastly, we
found that AIP1-GFP was enriched in a variety of developing
epithelial tissues including embryonic ectoderm undergoing
gastrulation, follicle epithelium, pupal eye epithelia undergoing
rhabdomere morphogenesis, larval wing disc epithelium and
Malpighian tubule (supplementary material Fig. S4C-G). These
data suggest that AIP1 could be an actin dynamics promoter that
specifically regulates AJ function during epithelial morphogenesis.
Cofilin is required for AJ remodeling and its
overexpression rescues AIP1-deficient AJ defects
To further determine whether cofilin plays a role in AJ remodeling
in ommatidial preclusters and clusters, we examined the eye disc
phenotype of mutants in twinstar (tsr), the gene that encodes
Fig. 6. AIP1 localizes in the apical region of preclusters and
clusters. (A-A⵮) Low magnification view of distribution patterns of
AIP1-GFP, F-actin and Arm in a larval eye disc. The yellow dotted line
marks the position of the cross-section, which is shown beneath each
panel. Arrow points to the MF. (B-C⵮) High magnification views
showing that AIP1 is enriched together with F-actin underlying the AJs
of preclusters (B-B⵮) and that AIP1 colocalizes with F-actin in the AJ
region of more mature clusters (C-C⵮). Cofilin displays a diffuse staining
pattern in the apical plane and does not show enrichment in the
clusters or preclusters (see supplementary material Fig. S5B-B⬙). Scale
bars: 10m in A-A⵮; 5m in B-C⵮.
Development 139 (19)
Drosophila cofilin. Since tsrnull results in cell lethality and
precludes the use of mosaic clones, we utilized two previously
reported temperature-sensitive alleles of tsr for functional analysis
(Blair et al., 2006). At the non-permissive temperature at 29°C, the
tsrV27Q/tsr139 allelic combination produced similar ectopic F-actin
and AJ remodeling defects (30.6% of third instar eye discs
displayed defects, n36) to those resulting from flr loss of function
(Fig. 7C-D⬙). Furthermore, we generated GFP-labeled clones
expressing tsr RNAi to knockdown tsr function using a flip-out
method, and the resulting F-actin and AJ phenotypes were similar
to those of tsr mutants. In the tsr mutant or knockdown eye discs,
the various stages of precluster formation (including rosettes, arcs
and five-cell preclusters) were not discernible in their distinct forms
within and immediately posterior to the MF (Fig. 7C;
supplementary material Fig. S6A-A⬙), and the more mature
ommatidial clusters displayed highly disorganized AJs and a lack
of apical constriction (Fig. 7D; supplementary material Fig. S6BB⬙), similar to the flr mutant. Furthermore, F-actin levels were
strongly increased in both preclusters and clusters, and a crosssectional view showed that the strong F-actin accumulation
occurred both apically and basally, which differs from the apical
accumulation found in the flr mutant (Fig. 7C⬘-D⬘; supplementary
material Fig. S6A⬘-B⬘). These results suggest that, like AIP1, the
actin disassembly function of cofilin is also required for AJ
remodeling. Interestingly, mutation in slingshot (ssh; which
encodes a cofilin phosphatase and activator) or capt (act-up; which
encodes a cofilin activator that recycles cofilin and actin for
depolymerization and polymerization, respectively) each caused AJ
defects and F-actin accumulation in preclusters and clusters
(supplementary material Fig. S6C-F⬙), consistent with previous
reports (Benlali et al., 2000; Corrigall et al., 2007; Rogers et al.,
2005).
To test whether tsr genetically interacts with flr in precluster and
cluster formations, we overexpressed the tsr+ transgene to rescue
the flr mutant defects. Using flip-out, we generated clones
expressing UAS-flr RNAi (together with UAS-lacZ), and the
resulting phenotypes of increased F-actin level and disorganized
AJs in precluster and cluster cells were the same as those of flr
mutant clones (Fig. 7E-F⬘). Strikingly, overexpressing the tsr+
transgene in these flr knockdown clones almost completely rescued
the ectopic F-actin and AJ defects (Fig. 7G-H⬘). In fact, the
previously increased F-actin level in the apical plane was
suppressed below the wild-type level present in the adjacent
tissues, while the morphology of preclusters and clusters appeared
normal (Fig. 7G-G⵮), suggesting that an increase in cofilinmediated actin dynamics is not detrimental to AJ remodeling. To
determine whether cofilin physically interacts with AIP1 in vivo,
an immunoprecipitation assay was performed using tissue extracts
from third instar flrGFP larvae. The result showed that GFP-tagged
AIP1 could be immunoprecipitated with cofilin in the extract (Fig.
1H), indicating a physical interaction between Drosophila cofilin
and AIP1.
Interestingly, we observed a twofold (2.0±0.1, n6) increase
of cofilin levels in the apical region within the flr RNAi clones
posterior to the MF (Fig. 7E⬙,I; supplementary material Fig.
S6H⬘), and this increase was not uniform in the clones and was
more prominent within clusters in the more posterior region.
However, no cofilin or F-actin increase or AJ defects were
detected in flr RNAi clones anterior to the MF or in the larval
wing disc epithelium (Fig. 7J-K⬙), where much less intercellular
movement and AJ remodeling were apparent. These results
suggest that differentiating epithelial cells undergoing robust
DEVELOPMENT
3568 RESEARCH ARTICLE
Actin dynamics and adherens junction remodeling
RESEARCH ARTICLE 3569
morphogenesis and AJ remodeling require a high level of actin
dynamics and have a mechanism to sense ectopic F-actin (lack
of actin dynamics), leading to elevation of the cofilin level in the
AJ region to depolymerize the ectopic and less dynamic actin
filaments present there. This twofold increase of cofilin activity
was however insufficient to make up for the loss of AIP1
function. The complete rescue of AJ defects and F-actin increase
achieved by a tenfold increase in cofilin levels (10.3±0.6, n5;
Fig. 7G⬙,I) due to tsr+ transgene overexpression suggests that
AIP1 can strongly enhance the actin disassembly activity of
cofilin in the AJ region.
DISCUSSION
Here, we demonstrate an essential role for the actin disassembly
factors AIP1 and cofilin in promoting the actin cytoskeleton
dynamics underlying AJs, which is crucial for AJ remodeling
during epithelial morphogenesis. Based on data from the
distribution patterns and the genetic and physical interaction
experiments, AIP1 appears to specifically act in the apical region
to enhance cofilin-mediated actin dynamics. Such a high level of
actin dynamics is crucial for AJ remodeling, as demonstrated by
Jasp treatment, which phenocopied the flr or tsr AJ defects within
and posterior to the MF. However, the distribution of cofilin is
DEVELOPMENT
Fig. 7. tsr mutations result in AJ defects and tsr+ overexpression rescues flr AJ defects. (A-D⬙) tsrV27Q/tsr139 results in AJ defects in the
preclusters (C) and clusters (D), as compared with the wild-type control (A,B). F-actin levels are also increased in tsr mutants (C⬘,D⬘), as compared
with the control (A⬘,B⬘); corresponding images (C⬘,A⬘; D⬘,B⬘) are taken at same confocal settings for intensity comparison. Arrow indicates MF;
arrowhead indicates a cluster with prominent AJ defects. (E-F⬘) flr RNAi clones marked by UAS-GFP and UAS-lacZ coexpression (E,F) display
increased levels of F-actin and cofilin staining (E-E⵮) and disrupted Arm staining in the AJs (F,F⬘) posterior to the MF. (G-H⬘) Overexpression of the
tsr+ transgene in the flr RNAi clones (marked by GFP in G,H) completely suppresses ectopic F-actin and AJ disruption defects (G⬘,H⬘). (I)Increase in
cofilin level in flr RNAi or flr RNAi + tsr+ overexpression clones calculated as the ratio of the fluorescence intensity (FI) of clones posterior to the MF
versus the FI of adjacent wild-type tissues posterior to the MF. The fluorescence and area of the clones or adjacent tissues were measured in ImageJ
to calculate respective FIs; data were analyzed using Student’s t-test. Error bars indicate s.e.m. of five or six eye discs; ***P<0.001. Note that images
in E⬙,G⬙ are not taken at the same settings. (J-K⬙) flr RNAi clones anterior to the MF (J-J⬙) or in the wing disc (K-K⬙) show no increase in the cofilin
level or AJ defects. Scale bars: 10m.
diffuse and it seems to provide a basal level of actin dynamics,
which might be generally required for multiple cellular processes
but is not sufficient for AJ remodeling. Recent studies have shown
that myosin II and Ssh are enriched in the apical region of
developing preclusters and clusters, similar to the AIP1 pattern
(Escudero et al., 2007; Rogers et al., 2005). Thus, evidence
suggests that the apical region of preclusters and clusters
constitutes a distinct zone that contains activators of cofilin to
effect a much enhanced rate of actin disassembly. Indeed, we and
others have found that strong AJ defects in preclusters and clusters
are caused by mutations in ssh or capt (Benlali et al., 2000;
Corrigall et al., 2007; Rogers et al., 2005) (supplementary material
Fig. S7), each of which encodes a cofilin activator.
A fundamental question in the field of epithelial morphogenesis
and AJ remodeling is how cells both maintain general cell adhesion
essential for their tight association and overall epithelial integrity
and allow intercellular movement to take place within cell sheets.
Results from our study support a model in which enhanced actin
depolymerizing function is essential to allow these two seemingly
opposing processes to take place simultaneously. First, active
polymerization in the apical region would ensure a constant supply
and a certain level of F-actin to be always available underlying AJs.
Such a level of F-actin is essential for the general cell adhesion
mediated by the E-cad–-catenin–-catenin complex connecting to
the actin network, as blocking endogenous actin polymerization
with Lat-A caused severe disruption of E-cad-GFP localization in
the AJs within the MF and preclusters, often accompanied by
collapse of epithelial integrity around this region. Indeed, the Jasp
recovery assay demonstrates that actin polymerization appeared to
be most robust in this MF-proximal region, and we also found that
the actin polymerization factor acin polymerization factor Arp2
(Arp14D – FlyBase; an essential subunit of the Arp2/3 complex)
is upregulated posterior to the MF and is enriched in the apical/AJ
plane, displaying a similar distribution pattern to that of AIP1
(supplementary material Fig. S5A-A⬙). Arp2/3 has been implicated
to affect AJ remodeling through its direct interaction with -catenin
and the underlying actin network, or through its promotion of Ecad endocytosis (Georgiou et al., 2008; Harris and Tepass, 2010).
However, what role Arp2/3 plays during AJ remodeling in the eye
epithelium remains to be elucidated.
Second, AIP1, and probably other cofilin activators, provide
much enhanced actin depolymerization underlying AJs to make the
highly polymerized F-actin more dynamic or treadmilling at a
faster pace. A rapidly turning over actin network has been shown
to be essential for lateral mobility of E-cad clusters in AJs of
embryonic epithelium, and excessive polymerization restricts the
mobility (Cavey et al., 2008). Consistently, we showed that Jasp
treatment and flr mutation resulted in a significant reduction of the
E-cad-GFP turnover rate in the precluster AJs and in a failure of
precluster progenitor cells to undergo cell rearrangement. It is
interesting to note that the AJs of preclusters within the four to five
columns posterior to the MF displayed the most robust actin
polymerization and were sensitive to both Jasp and Lat-A,
suggesting that both actin polymerization and depolymerization
need to be at high levels (i.e. fast actin treadmilling) to effect fast
AJ remodeling in this morphogenetically active region of the eye
disc.
Our live imaging study shows that precluster formation involves
a series of stereotypical steps: from a large rosette of ~20 cells to
an arc of about six or seven cells and finally a five-cell precluster,
which is largely consistent with the description of the stages of
precluster formation inferred from staining of fixed samples (Wolff
Development 139 (19)
and Ready, 1991). However, live imaging revealed new details of
the dynamics of AJ remodeling. We found that this patterned
morphogenetic program always involved a transient intermediate
stage of a six-cell rosette, with the five R cells and one M cell
sharing a common AJ vertex. Such a rosette is crucial for R3 and
R4 to establish AJ contact with each other and for the M cell to be
excluded from contact with R8, R2 and R5. In the flr mutant, these
six-cell rosettes were rarely observed during precluster formation.
Interestingly, similar kinds of multicellular rosette structures with
a common vertex have been described in various epithelial
morphogenetic systems including Drosophila embryonic ectoderm
and in gastrulating and neurulating chick embryos undergoing
primitive streak formation and neural plate invagination,
respectively (Blankenship et al., 2006; Nishimura and Takeichi,
2008; Wagstaff et al., 2008), and blocking the function of the
actomyosin network can prevent the formation of rosettes in these
systems. Thus, our findings support the notion that the rosette-like
structure could be a highly conserved intermediate form that is
crucial for epithelial morphogenesis in general.
Acknowledgements
We thank Frank Laski for providing fly strains and critical comments on the
manuscript; James Bamburg for antibody provision; and the Bloomington
Drosophila Stock Center, Vienna Drosophila RNAi Center and Drosophila
Genetic Resource Center for fly strains.
Funding
This work is supported by grants from the National Natural Sciences
Foundation of China [90608018] and Ministry of Science and Technology
[2007CB947101, 2006CB943503] to J.C.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.079491/-/DC1
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DEVELOPMENT
Actin dynamics and adherens junction remodeling